The introduction of cloned nucleotide sequences into mammalian cells has greatly facilitated the study of the control and function of various eukaryotic genes. Mammalian cells provide an environment conducive to appropriate protein folding, post translational processing, feedback control, protein-protein interactions, and other eukaryotic protein modifications such as glycosylation and oligomerization. Thus, a number of expression vectors have been developed which allow the expression of a polypeptide in a mammalian cell.
The typical mammalian expression vector will contain (1) regulatory elements, usually in the form of a viral promoter or enhancer sequences; (2) a multicloning site, usually having specific enzyme restriction sites to facilitate the insertion of a DNA fragment with the vector; and (3) sequences responsible for intron splicing and polyadenylation of mRNA transcripts. Generally, sequences facilitating the replication of the vector in both bacterial and mammalian hosts and a selection marker gene which allows selection of transformants in bacteria are also included. The bacterial elements, or in some cases phage elements, are included to provide the option of further analyzation of the nucleic acid inserts amplified and isolated from the bacteria or phage.
In the past, the insertion of a heterologous nucleic acid (insert) into the multicloning site of a mammalian expression vector has generally been accomplished by one of two methods. In the first method, the insert is cut out of a bacterial expression vector and ligated into the mammalian expression vector. In the second method, often called "TA cloning", special ends are generated on the insert by PCR such that the modified insert can be put into the mammalian expression vector. Each of these methods requires a number of steps including enzymatic reactions which can be labor intensive and unreliable. Moreover, cloning efficiency drops significantly as the size of the insert increases.
Another method used for inserting a heterologous nucleic acid (insert) into an expression vector takes advantage of yeast's high efficiency at homologous recombination in vivo. In this method, a nucleic acid fragment flanked by 5' and 3' homologous regions is co-introduced into a yeast with a vector which has regions identical to the 5' and 3' regions flanking the fragment. The yeast efficiently homologously recombines such that the fragment inserts into the region of the vector flanked by the before-mentioned 5' and 3' regions. H.a., et al., Plasmid, 38:91-96 (1997), incorporated herein. Unfortunately, yeast are the only organisms able to efficiently recombine so as to insert heterologous nucleic acids into a vector. Therefore, to date, there is not an efficient method or means to transfer inserts into a specific region of a vector used for expression in mammalian cells.
Accordingly, it is an object of the invention to provide compositions and methods useful in facilitating the insertion of a heterologous nucleic acid into a vector which can express the heterologous nucleic acid in at least a mammalian cell.
Moreover, it is the object of this invention to provide a shuttle vector and methods of use which allow replication of the shuttle vector at least in yeast and which allow expression in at least a mammalian cell.